The enteric nervous system (ENS) is remarkable for its phenotypic diversity and regulation of complex enteric behaviors independently of CNS input. Research into the pathogenesis of the congenital aganglionosis of Hirschsprung's disease (HSCR) has provided critical information about genes and molecules that act early in development to regulate colonization of the bowel by enteric neural crest-derived cells (ENCDC) and neurogenesis. Less is known about the later acting genes or molecules, which contribute to enteric neuronal phenotypic diversity. The current proposal is designed to develop an understanding of mechanisms that underlie the acquisition of this diversity, which when abnormal, disturb GI motility without giving rise to HSCR. We propose to test the hypothesis that neurotransmitters of early-born neurons, which co-exist with still-plastic dividing precursors, influence the fates of uncommitted ENCDC and, consequently, the late-born neurons that develop from them. Altered activity of early developing neurons that affects subsequent neuronal development might account for the long-lasting abnormal motility that have been observed in animals after neonatal irritation of the gut or maternal separation, as well as reports in humans with functional GI disorders of a history of infection, memories of abdominal pain during childhood, or a history of stress and/or abuse. The ENCDC that give rise to serotonergic neurons, which are among the first to born in the ENS, are transiently catecholaminergic (TC) and express the norepinephrine transporter (NET). Genetic NET deletion (NETKO) interferes with development of enteric serotonergic neurons. Neuronal 5-HT biosynthesis depends on tryptophan hydroxylase 2 (TPH2) while that in enterochromaffin cells depends on TPH1. The ENS of mice lacking neuronal 5-HT (TPH2KO), but not that of mice lacking mucosal 5-HT (TPH1KO), is deficient in total and dopaminergic neurons, although brain dopamine is normal. 5-HT thus promotes enteric neurogenesis/survival and the differentiation of enteric, but not CNS, dopaminergic neurons. In addition to 5-HT, the newly discovered, cerebral dopaminergic neurotrophic factor (CDNF) also contributes to ENS dopaminergic development. We will test hypotheses that NE (through NET) and 5-HT affect respectively, development of early-born and late-born enteric neurons. We propose that 5-HT is necessary for development of of late-born neurons, but does not, by itself, specify their phenotype, for example, to be dopaminergic. That requires additional factor(s), such as CDNF. Specifically, we will use transgenic mice and pharmacological tools to analyze the roles that norepinephrine and NET play in development/survival of enteric serotonergic neurons as well as the roles that enteric neuronal 5-HT and CDNF play in development/survival of dopaminergic and other late-born enteric neurons. We will also now analyze, in vivo and in vitro, the motility of mutant and wild-type gut to test the idea that the ENS defects that we identify in ! the mutant mice disturb intestinal tranit and motor patterns in adults despite the absence of aganglionosis. PUBLIC HEALTH RELEVANCE: No other component of the nervous system outside of the brain or spinal cord is as large, complex, or independent as the intrinsic innervation of the gut. Prior research on development of this innervation has been motivated by the need to understand the pathophysiology of Hirschsprung's disease, in which aggregates of nerve cells (ganglia) fail to arise in various length of terminal intestine. As a result, much more is known about the functions of early-acting genes that determine whether or not nerve cells develop in the bowel than about late-acting genes that determine whether nerve cells that develop do so in appropriate numbers and types. We have found that newly born nerve cells coexist in the primordial nervous system of the gut with still- dividing and plastic precursors. As a result, the activity of the early arising nerve clls can alter the local environment and thus the fates of later developing nerve cells. That activity, in turn, is potentially responsive to many types of input, including infection, inflammation, stres, and psychological trauma. Neuronal regulation of neuronal development would thus provide a common pathway for those stimuli to shape the nature of a still plastic and developing intrinsic gastrointestinal innervation. We now propose to test these concepts, investigating specifically the early role of the norepinephrine transporter in the development of neurons that use serotonin as their transmitter, the later role of serotonin in the development of neurons that use dopamine as their transmitter, and finally the interaction of serotonin with a newly-discovered cerebral dopaminergic neurotrophic factor that is expressed in the gut. The work, which now includes functional studies of the motility of mutant bowel, will provide insight into the developmental origins of functional gastrointestinal disorders as well as Parkinson's disease, which affects the gut, often before it affects the brain.